RESUMO
Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the aging process affects the response to injury of cholangiocytes. The aim of this study was to identify molecular pathways associated with cholangiocyte aging and to determine their effects in the biological response to injury of biliary cells. A panel of microRNAs (miRs) involved in aging processes was evaluated in cholangiocytes of young and old mice (2 months and 22 months of age, respectively) and subjected to a model of sclerosing cholangitis. Intracellular pathways that are common to elevated miRs were identified by in silico analysis. Cell proliferation and senescence were evaluated in Twinfilin-1 (Twf1) knocked-down cells. In vivo, senescence-accelerated prone mice (Samp8, a model for accelerated aging), Twf1-/- , or their respective controls were subjected to DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine). Cholangiocytes from DDC-treated mice showed up-regulation of a panel of aging-related miRs. Twf1 was identified by in silico analysis as a common target of the up-regulated miRs. Twf1 expression was increased both in aged and diseased cholangiocytes, and in human cholangiopathies. Knock-down of Twf1 in cholangiocytes reduced cell proliferation. Senescence and senescence-associated secretory phenotype marker expression increased in Twf1 knocked-down cholangiocytes following pro-proliferative and pro-senescent (10-day lipopolysaccharide) stimulation. In vivo, Samp8 mice showed increased biliary proliferation, fibrosis, and Twf1 protein expression level, whereas Twf1-/- had a tendency toward lower biliary proliferation and fibrosis following DDC administration compared with control animals. Conclusion: We identified Twf1 as an important mediator of both cholangiocyte adaptation to aging processes and response to injury. Our data suggest that disease and aging might share common intracellular pathways.
Assuntos
Senescência Celular/genética , Colangite Esclerosante/patologia , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Envelhecimento/genética , Animais , Sistema Biliar/patologia , Proliferação de Células/genética , Células Cultivadas , Colangite Esclerosante/genética , Modelos Animais de Doenças , Humanos , Camundongos , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND AIMS: Liver X receptors (LXRs) exert anti-inflammatory effects even though their hepatic activation is associated with hypertriglyceridemia and hepatic steatosis. Selective induction of LXRs in the gut might provide protective signal(s) in the aberrant wound healing response that induces fibrosis during chronic liver injury, without hypertriglyceridemic and steatogenic effects. METHODS: Mice with intestinal constitutive LXRα activation (iVP16-LXRα) were exposed to intraperitoneal injection of carbon tetrachloride (CCl4 ) for 8 weeks, and in vitro cell models were used to evaluate the beneficial effect of high-density lipoproteins (HDL). RESULTS: After CCl4 treatment, the iVP16-LXRα phenotype showed reduced M1 macrophage infiltration, increased expression M2 macrophage markers, and lower expression of hepatic pro-inflammatory genes. This anti-inflammatory effect in the liver was also associated with decreased expression of hepatic oxidative stress genes and reduced expression of fibrosis markers. iVP16-LXRα exhibited increased reverse cholesterol transport in the gut by ABCA1 expression and consequent enhancement of the levels of circulating HDL and their receptor SRB1 in the liver. No hepatic steatosis development was observed in iVP16-LXRα. In vitro, HDL induced a shift from M1 to M2 phenotype of LPS-stimulated Kupffer cells, decreased TNFα-induced oxidative stress in hepatocytes and reduced NF-kB activity in both cells. SRB1 silencing reduced TNFα gene expression in LPS-stimulated KCs, and NOX-1 and IL-6 in HepG2. CONCLUSIONS: Intestinal activation of LXRα modulates hepatic response to injury by increasing circulating HDL levels and SRB1 expression in the liver, thus suggesting this circuit as potential actionable pathway for therapy.
Assuntos
Células de Kupffer , Fígado , Animais , HDL-Colesterol , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Microbial products are thought to influence the progression of cholangiopathies, in particular primary sclerosing cholangitis (PSC). Inflammasomes are molecular platforms that respond to microbial products through the synthesis of proinflammatory cytokines. We investigated the role of inflammasome activation in cholangiocyte response to injury. Nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing protein 3 (Nlrp3) expression was tested in cholangiocytes of normal and cholestatic livers. Effects of Nlrp3 activation induced by incubation with lipopolysaccharide and ATP was studied in vitro in normal and siRNA-Nlrp3 knocked-down cholangiocytes. Wild-type and Nlrp3 knockout (Nlrp3-/-) mice were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a model of sclerosing cholangitis) for 4 weeks. Nlrp3 and its components were overexpressed in cholangiocytes of mice subjected to DDC and in patients affected by PSC. In vitro, Nlrp3 activation stimulated expression of Il-18 but not of Il-1ß and Il-6. Nlrp3 activation had no effect on cholangiocyte proliferation but significantly decreased the expression of Zonulin-1 and E-cadherin, whereas Nlrp3 knockdown increased the permeability of cholangiocyte monolayers. In vivo, the DDC-stimulated number of cytokeratin-19-positive cells in the liver of wild-type animals was slightly reduced in Nlrp3-/- mice, and expression of E-cadherin was reestablished. In conclusion, Nlrp3 is expressed in reactive cholangiocytes, in both murine models and patients with PSC. Activation of Nlrp3 leads to synthesis of proinflammatory cytokines and influences epithelial integrity of cholangiocytes.
Assuntos
Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Interleucina-18/biossíntese , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Humanos , Immunoblotting , Inflamassomos/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram-negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high-fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut-sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL- or HFD-treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram-negative versus Gram-postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram-negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD-treated mice; liver damage was further enhanced by transplantation of selected Gram-negative bacteria obtained from cecum content of HFD+BDL-treated mice. CONCLUSIONS: Dietary habits, by increasing the percentage of intestinal Gram-negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD.
Assuntos
Disbiose/complicações , Cirrose Hepática Experimental/etiologia , Animais , Translocação Bacteriana , Tetracloreto de Carbono/toxicidade , Dieta Hiperlipídica , Trato Gastrointestinal/microbiologia , Inflamassomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/fisiologiaRESUMO
UNLABELLED: The activation of the biliary stem-cell signaling pathway hairy and enhancer of split 1/pancreatic duodenal homeobox-1 (Hes-1/PDX-1) in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and ductal cell neogenesis. PDX-1-dependent activation of Ngn-3 initiates the differentiation program by inducing microRNA (miR)-7 expression. Here we investigated the role Ngn-3 on cholangiocyte proliferation. Expression levels of Ngn-3 and miR-7 isoforms were tested in cholangiocytes from normal and cholestatic human livers. Ngn-3 was knocked-down in vitro in normal rat cholangiocytes by short interfering RNA (siRNA). In vivo, wild-type and Ngn-3-heterozygous (+/-) mice were subjected to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding (a model of sclerosing cholangitis) or bile duct ligation (BDL). In the liver, Ngn-3 is expressed specifically in cholangiocytes of primary sclerosing cholangitis (PSC) patients and in mice subjected to DDC or BDL, but not in normal human and mouse livers. Expression of miR-7a-1 and miR-7a-2 isoforms, but not miR-7b, was increased in DDC cholangiocytes compared to normal ones. In normal rat cholangiocytes, siRNA against Ngn-3 blocked the proliferation stimulated by exendin-4. In addition, Ngn-3 knockdown neutralized the overexpression of insulin growth factor-1 (IGF1; promitotic effector) observed after exposure to exendin-4, but not that of PDX-1 or VEGF-A/C. Oligonucleotides anti-miR-7 inhibited the exendin-4-induced proliferation in normal rat cholangiocytes, but did not affect Ngn-3 synthesis. Biliary hyperplasia and collagen deposition induced by DDC or BDL were significantly reduced in Ngn-3(+/-) mice compared to wild-type. CONCLUSION: Ngn-3-dependent activation of miR-7a is a determinant of cholangiocyte proliferation. These findings indicate that the reacquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ductos Biliares/fisiopatologia , Proliferação de Células/fisiologia , Colestase/fisiopatologia , MicroRNAs/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transdução de Sinais/fisiologia , Lesão Pulmonar Aguda/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colestase/metabolismo , Colestase/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Exenatida , Humanos , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos/farmacologia , Peptídeos/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Peçonhas/metabolismoRESUMO
Semaphorin7A (SEMA7A) is a membrane-anchored protein involved in immune and inflammatory responses, exerting an effect on pulmonary fibrosis. Thus, we aimed to investigate the role of SEMA7A in hepatic fibrosis. Liver injury was induced in vivo by carbon tetrachloride i.p. injection or bile duct ligation in wild-type and SEMA7A knockout (KO) mice. Human and mouse liver samples and primary mouse hepatic cell populations were used for Western blot analysis, quantitative real-time RT-PCR, and immunohistochemistry. SEMA7A is highly expressed in hepatic stellate cells (HSCs). The expression of SEMA7A and its receptor ß1-integrin subunit increase during liver injury and in activated HSCs. Transforming growth factor ß-stimulated HSCs showed increased expression of SEMA7A in a SMAD2/3-independent manner, leading to increased expression of fibrogenic and inflammation markers. This pattern was significantly blunted in SEMA7A KO HSCs. Overexpression of SEMA7A in HSCs showed increased fibrogenic and inflammation markers expression. In vivo, SEMA7A KO mice treated with carbon tetrachloride and bile duct ligation developed reduced fibrosis versus wild-type mice. Moreover, SEMA7A expression increased in liver samples of patients with fibrosis versus healthy controls. SEMA7A was expressed in the liver and was increased in the course of liver fibrosis, both in mice and in humans. SEMA7A was mainly expressed in HSCs with respect to other cell types in the liver and plays a critical role in regulating fibrosis.
Assuntos
Antígenos CD/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Semaforinas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/patologia , Humanos , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismoRESUMO
BACKGROUND & AIMS: Cholangiocyte proliferation plays a role in the progression of cholangiopathies, in particular in primary sclerosing cholangitis. The mechanisms regulating cholangiocyte proliferation are still undefined. Pancreatic Duodenal Homeobox protein 1 (PDX-1) is expressed by reactive cholangiocytes. In the adult pancreas, PDX-1 regulates the proliferative response to injury of ductal cells. Its effects can be counteracted by Hairy and enhancer of split 1 (Hes-1). We aimed at studying whether PDX-1/Hes-1 interactions regulate cholangiocyte proliferation in response to injury. METHODS: The effect of the loss of PDX-1 on cholangiocyte proliferation was studied in vitro. In vivo PDX-1-heterozygous (+/-) mice were subjected to either DDC feeding (a model of sclerosing cholangitis) or to bile duct ligation (BDL). PDX-1/Hes-1 interactions on cell proliferation were determined by exposure to All-trans Retinoic Acid (At-RA), an inductor of Hes-1. RESULTS: In vitro, cholangiocyte proliferation was undetectable in cells pre-treated with PDX-1 siRNA. In vivo, increases in bile duct mass and collagen deposition observed after DDC feeding or BDL were significantly reduced in PDX-1(+/-) mice. Hes-1 expression is reduced in proliferating cholangiocytes; At-RA induced a dose-dependent increase in Hes-1 and a decrease in PDX-1 expression. At-RA neutralized the increases in PDX-1 expression and cell proliferation, both in vitro and in vivo in DDC mice. PDX-1 is overexpressed and Hes-1 downregulated in cholangiocytes isolated from PSC livers. CONCLUSIONS: Hes-1 downregulation allows PDX-1 to act as a major determinant of cholangiocyte proliferation in response to cholestatic injury. These findings provide novel mechanistic insights into the pathophysiology of cholangiopathies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Colangite Esclerosante/etiologia , Colangite Esclerosante/patologia , Proteínas de Homeodomínio/metabolismo , Transativadores/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sistema Biliar/lesões , Proliferação de Células , Células Cultivadas , Colangite Esclerosante/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Heterozigoto , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição HES-1RESUMO
BACKGROUND: Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated. AIM: To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution METHODS: HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats. RESULTS: In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype. CONCLUSIONS: ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution.
Assuntos
Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Fibrose/fisiopatologia , Células Estreladas do Fígado/fisiologia , Animais , Apoptose/efeitos dos fármacos , Ductos Biliares/cirurgia , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Caspase 8/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Ligadura , Fígado/patologia , RNA Interferente Pequeno/genética , Ratos , Tapsigargina/farmacologia , Tunicamicina/farmacologiaRESUMO
BACKGROUND/AIMS: High-fat dietary intake and low physical activity lead to insulin resistance, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Recent studies have shown an effect of glucagon-like peptide-1 (GLP-1) on hepatic glucose metabolism, although GLP-1 receptors (GLP-1r) have not been found in human livers. The aim of this study was to investigate the presence of hepatic GLP-1r and the effect of exenatide, a GLP-1 analogue, on hepatic signalling. METHODS: The expression of GLP-1r was evaluated in human liver biopsies and in the livers of high-fat diet-treated rats. The effect of exenatide (100 nM) was evaluated in hepatic cells of rats fed 3 months with the high-fat diet. RESULTS: GLP-1r is expressed in human hepatocytes, although reduced in patients with NASH. Similarly, in rats with NASH resulted from 3 months of the high-fat diet, we found a decreased expression of GLP-1r and peroxisome proliferator-activated receptor γ (PPARγ), and reduced peroxisome proliferator-activated receptor α (PPARα) activity. Incubation of hepatocytes with exenatide increased PPARγ expression, which also exerted an insulin-sensitizing action by reducing JNK phosphorylation. Moreover, exenatide increased protein kinase A (PKA) activity, Akt and AMPK phosphorylation and determined a PKA-dependent increase of PPARα activity. CONCLUSIONS: GLP-1 has a direct effect on hepatocytes, by activating genes involved in fatty acid ß-oxidation and insulin sensitivity. GLP-1 analogues could be a promising treatment approach to improve hepatic insulin resistance in patients with NAFLD/NASH.
Assuntos
Gorduras na Dieta/metabolismo , Fígado Gorduroso/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Fígado/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Glucagon/agonistas , Transdução de Sinais/efeitos dos fármacos , Peçonhas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Biópsia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Exenatida , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Receptor do Peptídeo Semelhante ao Glucagon 1 , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Resistência à Insulina , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica , Oxirredução , PPAR alfa/metabolismo , PPAR gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Fatores de TempoRESUMO
BACKGROUND & AIMS: Reactive cholangiocytes acquire a neuroendocrine-like phenotype, with synthesis and local release of neuropeptides and hormones. The mechanism that drives such phenotypical changes is still undefined. Pancreatic Duodenal Homeobox-1 (PDX-1) is a transcription factor required for pancreatic development, that sustains pancreatic beta-cell response to injury and insulin synthesis. PDX-1 induces neuroendocrine-like transition of pancreatic ductal cells. Cholangiocyte response to injury is modulated by Glucagon-Like Peptide-1 Receptor (GLP-1R), which, in the pancreas, activates PDX-1. We wanted to verify whether PDX-1 plays any role in cholangiocyte neuroendocrine-like transdifferentiation in response to injury. METHODS: PDX-1 expression was assessed in cholangiocytes from normal and one week bile duct ligated (BDL) rats. Changes in PDX-1 expression and activation upon GLP-1R activation were then assayed. The effects of the lack of PDX-1 in cholangiocytes were studied in vitro by siRNA and in vivo by the employment of PDX-1-deficient (+/-) mice. RESULTS: BDL but not normal cholangiocytes express PDX-1. GLP-1R activation elicits, in a PI3K-dependent fashion, PDX-1 expression, together with its nuclear translocation. In vitro, GLP-1R-induced increases in VEGF and IGF-1 mRNA expression were blunted in cells with PDX-1 siRNA. In vivo, the VEGF and IGF-1 mRNA expression in the liver after one week BDL was markedly reduced in PDX-1-deficient mice, together with reduced bile duct mass. CONCLUSIONS: In response to injury, reactive cholangiocytes de novo express PDX-1, the activation of which allows cholangiocytes to synthesize IGF-1 and VEGF. These findings suggest that PDX-1 drives the acquisition of the neuroendocrine-like phenotype by cholangiocytes in response to cholestatic injury.
Assuntos
Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proteínas de Homeodomínio/genética , Transativadores/genética , Animais , Diferenciação Celular , Transdiferenciação Celular , Expressão Gênica , Humanos , Camundongos , RatosRESUMO
This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.
Assuntos
Família 4 do Citocromo P450/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR gama/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , alfa-Tocoferol/metabolismo , Animais , Família 4 do Citocromo P450/genética , Dieta da Carga de Carboidratos/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Dieta Ocidental/efeitos adversos , Ácidos Graxos não Esterificados/efeitos adversos , Frutose/efeitos adversos , Células Hep G2 , Humanos , Hidroxilação , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Ácido Oleico/efeitos adversos , PPAR gama/genética , Ácido Palmítico/efeitos adversos , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Context: Contrasting data have been reported on the role of irisin, a novel myokine encoded by the fibronectin type III domain-containing protein 5 (FNDC5) gene, in nonalcoholic fatty liver disease (NAFLD) pathogenesis. We tested in patients with suspected nonalcoholic steatohepatitis (NASH) the association of FNDC5 variants, hepatic expression, and circulating irisin with liver damage (F2 to F4 fibrosis as main outcome). We also investigated whether irisin modulates hepatocellular fat accumulation and stellate cell activation in experimental models. Methods: We considered 593 consecutive patients who underwent liver biopsy for suspected NASH and 192 patients with normal liver enzymes and without steatosis. FNDC5 rs3480 and rs726344 genotypes were assessed by 5' nuclease assays. Hepatic irisin expression was evaluated in mice fed a high-fat diet or treated with CCl4. The effect of irisin was evaluated in fat-laden HepG2 hepatocytes and in hepatic stellate cells (HSCs). Results: In patients at risk for NASH [odds ratio (OR) = 0.64, 95% confidence interval (CI), 0.47 to 0.87; P = 0.005], and more so in the high-risk subgroup of those with impaired fasting glucose/diabetes (OR = 0.44, 95% CI, 0.26 to 0.74; P = 0.002), the rs3480 A>G variant was independently associated with protection from F2 to F4 fibrosis. Irisin is expressed in human activated HSC, where it mediated fibrogenic actions and collagen synthesis, and is overexpressed in NAFLD patients with F2 to F4 fibrosis and CCl4-treated mice. However, Irisin does not affect fat accumulation in HepG2 and is not induced by high-fat-diet-inducing NAFLD. Conclusions: The FNDC5 rs3480 variant is associated with protection from clinically significant fibrosis in patients with NAFLD, while irisin expression is correlated with the severity of NAFLD and may be involved in extracellular matrix deposition. These data suggest that irisin is involved in regulation of hepatic fibrogenesis.
Assuntos
Fibronectinas/genética , Fibronectinas/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/genética , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Animais , Tetracloreto de Carbono/toxicidade , Estudos de Casos e Controles , Dieta Hiperlipídica , Feminino , Predisposição Genética para Doença , Células Hep G2 , Humanos , Técnicas In Vitro , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Razão de Chances , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de DoençaRESUMO
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
RESUMO
Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A "multi-hit" theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3-/- and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3-/--HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3-/--HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.
Assuntos
Translocação Bacteriana/imunologia , Disbiose/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Translocação Bacteriana/efeitos dos fármacos , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Disbiose/tratamento farmacológico , Frutose/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Humanos , Imunidade Inata , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Hepatopatia Gordurosa não Alcoólica/etiologia , Permeabilidade , Fenótipo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH).We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1ß, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1ß transcription exclusively required LITAF expression/activity. Finally, IL-1ß levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH.In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1ß levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.
Assuntos
Citocinas/metabolismo , Células Estreladas do Fígado/metabolismo , Mediadores da Inflamação/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Citocinas/genética , Proteínas de Ligação a DNA , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/patologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Nucleares/genética , Fenótipo , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND AND AIMS: Endoscopic ultrasound-guided fine needle aspiration is routinely used in the diagnostic work up of pancreatic cancer but has a low sensitivity. Studies showed that Pancreatic Duodenal Homeobox-1 (PDX-1) is expressed in pancreatic cancer, which is associated with a worse prognosis. We aimed to verify whether the assessment of PDX-1 in endoscopic ultrasound-guided fine needle aspiration samples may be helpful for the diagnosis of pancreatic cancer. METHODS: mRNA of 54 pancreatic cancer and 25 cystic lesions was extracted. PDX-1 expression was assessed by Real-Time PCR. RESULTS: In all but two patients with pancreatic cancer, PDX-1 was expressed and was found positive in 7 patients with pancreatic cancer in which cytology was negative. The positivity was associated with a probability of 0.98 (95% CI 0.90-1.00) of having cancer and the negativity with one of 0.08 (95% CI 0.01-0.27). The probability of cancer rose to 1.00 (95% CI 0.97-1.00) for patients positive to both PDX-1 and cytology and fell to 0.0 (95% CI 0.00-0.15) in patients negative for both. CONCLUSIONS: PDX-1mRNA is detectable in samples of pancreatic cancer. Its quantification may be helpful to improve the diagnosis of pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/genética , Cistadenocarcinoma Mucinoso/genética , Cistadenocarcinoma Seroso/genética , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Idoso , Carcinoma Ductal Pancreático/diagnóstico , Estudos de Casos e Controles , Cistadenocarcinoma Mucinoso/diagnóstico , Cistadenocarcinoma Seroso/diagnóstico , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico , Pseudocisto Pancreático/diagnóstico , Pancreatite Crônica/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. AIM: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. METHODS: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. RESULTS: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1-3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. CONCLUSIONS: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.
Assuntos
Carcinoma Hepatocelular/etiologia , Modelos Animais de Doenças , Alimentos Formulados , Resistência à Insulina/fisiologia , Neoplasias Hepáticas/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Fatores Etários , Animais , Deficiência de Colina , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
BACKGROUND: Cholangiocarcinoma cells over-express oestrogen receptor-ß, which displays anti-proliferative and pro-apoptotic effects. AIM: To evaluate the effects of a newly developed and highly selective oestrogen receptor-ß agonist (KB9520) on experimental intrahepatic cholangiocarcinoma. METHODS: In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-ß silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-α and oestrogen receptor-ß negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-α) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested. RESULTS: In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-α and ß (HepG2) and in cells expressing only oestrogen receptor-α (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-ß expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis. CONCLUSIONS: KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-ß, suggesting that oestrogen receptor-ß selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Receptor beta de Estrogênio/agonistas , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Animais , Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Western Blotting , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Receptor beta de Estrogênio/biossíntese , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , RNA Neoplásico , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
Cholangiocytes are the epithelial cells that line the biliary tree; they are the target of chronic diseases termed cholangiopathies, which represent a daily challenge for clinicians, since definitive medical treatments are not available yet. It is generally accepted that the progression of injury in the course of cholangiopathies, and promotion and progression of cholangiocarcinoma are at least in part due to the failure of the cholangiocytes' mechanisms of adaptation to injury. Recently, several studies on the pathophysiology of the biliary epithelium have shed some light on the mechanisms that govern cholangiocyte response to injury. These studies provide novel information to help interpret some of the clinical aspects of cholangiopathies and cholangiocarcinoma; the purpose of this review is thus to describe some of these novel findings, focusing on their significance from a clinical perspective.
Assuntos
Doenças dos Ductos Biliares/fisiopatologia , Ductos Biliares/citologia , Sistema Nervoso Autônomo/fisiologia , Ácidos e Sais Biliares/fisiologia , Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares/inervação , Colestase/fisiopatologia , Citocinas/fisiologia , Células Epiteliais/fisiologia , Hormônios/fisiologia , Humanos , Inflamação/fisiopatologia , Neuropeptídeos/fisiologiaRESUMO
Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3-dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma.